title: Expression and purification of the recombinant lethal factor of Bacillus anthracis.
abstract: The structural gene for the 90-kDa lethal factor (LF) isolated from Bacillus anthracis was expressed as a fusion protein with six histidine residues in Escherichia coli. Expression of LF in E. coli under the transcriptional regulation of the T5 promoter yielded a soluble cytosolic protein with an apparent molecular mass of 90 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant LF reacted with anti-LF antibodies. The protein was purified to homogeneity by nickel nitrilotriacetic acid affinity chromatography and gel filtration on a Sephacryl S-200 column followed by anion exchange on a fast-performance liquid chromatograph with a Resource-Q column. The yield of purified LF from this procedure was 1.5 mg/liter. In solution, trypsin cleaved protective antigen bound to native and recombinant LF with comparable affinities. In macrophage lysis assays, native and recombinant LF exhibited identical potencies. The results suggest that large amounts of biologically active LF can be purified by this procedure.
authors.affiliation: Centre for Biotechnology, Jawahar Lal Nehru University, New Delhi, India.
authors.forename: P
authors.initials: P
authors.lastname: Gupta
authors.forename: S
authors.initials: S
authors.lastname: Batra
authors.forename: A P
authors.initials: AP
authors.lastname: Chopra
authors.forename: Y
authors.initials: Y
authors.lastname: Singh
authors.forename: R
authors.initials: R
authors.lastname: Bhatnagar
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011994:Recombinant Proteins; C030325:anthrax toxin